Our previous research illustrates that genetic muscular dystrophy of the chicken (line 307) is intrinsic to the muscle cell and is expressed in muscle cell cultures in the absence of innervation. Specifically, degradation of the 200,000 dalton subunit of myofibrillar myosin (MHC) and other major myofibrillar proteins is apparently accelerated by at least 50-75% in cell cultures prepared from the pectoralis muscle of twelve-day dystrophic embryos. These observations are consistent with previous reports of accelerated protein breakdown in dystrophic animals and indicate that aberrant proteolysis of myofibrillar proteins is a primary characteristic of the disease. Therefore, this project will investigate abnormalities in proteolytic systems in steady-state dystrophic muscle cell cultures, primarily focusing on myofibrillar proteins and employing specific protease inhibitors as probes for these abnormalities. The specific aims are: 1) To quantitate the specific radioactivity of precursor pool (3H) Leu in normal and dystrophic muscle cultures during pulse-labeling experiments. This information will enable us to calculate the absolute MHC breakdown rate in normal and dystrophic cells, since the experiments will be conducted in steady-state cultures where synthesis exactly equals breakdown. 2) To determine whether the observed abnormalities in MHC breakdown are also manifested in the metabolism of selected other myofibrillar and membrane proteins. These experiments will employ dual isotope labeling and two dimensional gel electrophoretic analysis of specific polypeptides. 3) In an attempt to identify the proteolytic systems responsible for breakdown of the major myofibrillar proteins, specific protease inhibitors (primarily of microbial origin) will be evaluated for their effects on protein breakdown in muscle cell cultures. Additionally, an endogenous inhibitor of muscle CAtt-activated protease will be inserted into muscle cultures in order to learn the true function of the protease. In conclusion, the ultimate medical goals of this research are to identify the abnormality protein breakdown that leads to severe muscle wasting in human muscular dystrophy, and to develop a suitable therapy by specifically inhibiting aberrant protein breakdown.